LRP1 is a member of the low-density lipoprotein receptor “LDLR”. LRP1 is a large protein of 4525 amino acids (600 kDa), which is cleaved by furin to produce two subunits of 515-(alpha) kD and 85-(β) kDa that remain non-covalently bound. LRP is expressed on most tissue types, but is primarily found in the liver. Other members of the low-density lipoprotein (LDL) receptor family include LDL-R (132 kDa); LRP2 (megalin, gp330); LRP/LRP1 and LRP1B (600 kDa); VLDL-R (130 kDa); LRP5; LRP6; apoER-2 (LRP-8, 130 kDa); Mosaic LDL-R (LR11, 250 KDa); and other members such as LRP3, LRP6, and LRP-7.
LRP1 is believed to be a multifunctional receptor. A binding repeat, resembling those found in the LDL receptor, is the molecular principle for the ability to bind a variety of ligands that were previously thought to be unrelated. These include the ligands lactoferrin, receptor associated protein (RAP), lipoprotein lipase, apoE, Factor VIII, beta-amyloid precursor, alpha-2-macroglobulin, thrombospondin 2 MMP-2 (matrix metalloproteinase-2), MPP-9-TIMP-1 (tissue inhibitor of matrix metalloproteinase-1); uPA (urokinase plasminogen activator):PAI-I (plasminogen activator inhibitor-1):uPAR (uPA receptor); and tPA (tissue plasminogen activator):PAI-1:uPAR, Pseudomonas exotoxin A, and human rhinovirus. See, Meilinger et al., FEBS Lett, 360:70-74 (1995). LRP1 is has the GenBank Accession No.: X 13916 and SwissProt Primary Accession No.: Q07954. Alternative names for the LRP1 gene/protein include: Low-density lipoprotein receptor-related protein 1 [precursor], LRP, Alpha-2-macroglobulin receptor, A2MR, Apolipoprotein E receptor, ApoER, CD91, LRP1 or A2MR.
An endoplasmic reticular chaperone protein, the receptor-associated protein (RAP), binds to complement repeat (CR) sequences within most LDLR. RAP assists in the folding of LDLR within the secretory pathway and antagonizes binding of all other known ligands to LDLR (Bu, (2001) Int Rev Cytol 209, 79-116). Despite the lack of detailed structural information on RAP, the association of RAP with the CR fold has been extensively characterized by a combination of receptor binding assays, calorimetry, and mutagenesis (Andersen, et al., (2001) Biochemistry 40, 15408-15417; Andersen, et al., (2000) J Biol Chem 275, 21017-21024; Migliorini, et al., (2003) J Biol Chem 278, 17986-17992; Neels, et al., (1999) J Biol Chem 274, 31305-31311; Horn, et al., (1997) J Biol Chem 272, 13608-13613)).
RAP is comprised of an array of three weakly homologous domains (Obermoeller, et al., (1997) J Biol Chem 272, 10761-10768). Each of these domains (d1, d2 and d3) has been shown to bind with varying affinity to pairs of immediately adjacent CR sequences within the LDLR ectodomains. Each of the effects of full-length RAP on LDLR, including facilitation of folding and inhibition of the binding of most other ligands (except α-2-macroglobulin), are recapitulated by RAPd3 alone. RAP d3 comprises amino acids 200-323 of mature Uniprot P30533 and amino acids 234-357 of precursor Uniprot P30533.
Hepatocytes are epithelial cells lining the vascular sinusoids of the liver. This cell type constitutes about 80% of total liver mass, providing the vast blood contact surface necessary for the function of the organ. Hepatocytes express large amounts of the low-density lipoprotein receptor-associated protein (LRP1), which participates in lipoprotein metabolism, specifically chylomicron remnant clearance (1,2,3), as well as uptake of other circulating proteins into the liver (2,4,5). Consistent with its physiological roles, LRP1 is a highly efficient ligand trafficking receptor, undergoing constitutive endocytosis with rapid internalization and recycling rates (6). Following internalization, LRP1 delivers bound cargo to the lysosome where the protein is rapidly degraded.
Hepatocellular carcinoma (HCC) originates from hepatocytes or their progenitors. HCC is the fifth most common cancer worldwide, the third most common cause of cancer-related death, and has shown increasing incidence in the U.S (7,8,9,10,11,12). The probability of developing hepatocellular carcinoma increases with viral infection (hepatitis B and C), alcoholism, smoking and obesity. Prognosis for this disease is poor, with a reported 5-year median survival rate of under 5%. Surgical resection, transplant and physical ablation are first choices for treatment, but only 5 to 10% of patients present with tumors suitable for these approaches. Tumor size, tumor dissemination within the liver, metastasis, diminished levels of organ function and high levels of recurrence limit the effectiveness of surgical intervention (13). Transarterial chemoembolism (a procedure in which the blood supply to a tumor is blocked (embolized) and chemotherapy is administered directly into the tumor) and intrahepatic chemotherapy (direct introduction of chemotherapy into liver tissue) have been shown to be helpful in some cases, but also give low overall rates of response (14,15). Systemic chemotherapy, such as adriamycin yields response rates of 15-20%, both because of the systemic toxicity of effective chemotherapeutics and tumor-cell resistance to the same (16,17). As the majority of HCC cases occur in patients whose liver function is already compromised, effective chemotherapeutic regimens, most of which result in some hepatoxicity, are often contraindicated due to insufficient hepatic reserve and the risk of fulminant liver failure. Targeted delivery of conjugated therapeutics, such as 90Yttrium, to liver after intravenous administration would significantly reduce the systemic toxicities associated with these drugs, reducing risk to the patient during treatment of HCC. One method of providing this targeted delivery could employ liver specific molecules, or ligands that bound to receptors on the liver with high-affinity, such as RAP.
Thus, there is a need in the art to further abate patient risk by selective tumor targeting of therapeutics to the liver using high-affinity ligands, such as RAP-dependent blood-borne delivery of chemotherapeutics or other agents to hepatocellular locations, to treat liver carcinoma or other liver diseases.